The use of DNA for diagnostics has seen a steady increase in recent years. The means of obtaining, isolating and analyzing the DNA have been extensively studied. A number of methods for the isolation of the DNA are extremely intrusive, such as the use of amniocenteses for the isolation of fetal blood, or biopsies to isolate genetic material associated with tumors for cancer diagnosis and monitoring. These methods are often painful and can lead to possible complications. Thus there exists a need to develop a less intrusive and safer way to isolate DNA for diagnostic uses.
It has recently been discovered that human urine contains sub microgram per milliliter amounts of DNA (Su et al., 2004). This DNA has been found to be comprised of two main types of DNA from two different sources. The larger species is generally greater than 1 kbp in size, and appears to be derived mainly from cells shed into the urine from the urinary tract. The second species is smaller, generally between 150 and 250 bp, and is mostly recovered from the supernatant. Researchers have found that this smaller species of urine DNA derives, at least in part, from the circulation.
It has been determined that some of this circulating DNA is a result of cell death. Upon cell death, cellular components are dismantled and usually phagocytosed by macrophages or neighboring cells. The nuclear DNA is often degraded by a range of enzymes, producing nucleosomes and their oligomers. In human adults, approximately 1011 cells die daily as a result of either disease or apoptosis. This corresponds to approximately 0.6 grams of DNA being released from the cells. It has been found that some of this DNA from dying cells escapes intracellular degradation and phagocytosis, and is able to circulate in the bloodstream. Then, some of this DNA from the bloodsteam is able to cross the kidney barrier and ends up in urine (Botezatu et al., 2000).
It is not currently known how small DNA can cross the kidney barrier. The nature of the cell-free DNA in blood has been extensively studied. Nucleosome-sized circulating DNA might originate from the internucleosomal cleavage of chromatin, a major hallmark of apoptosis. Malignant, benign, or even pre-neoplastic cells often proliferate at abnormal rates that are accompanied by an increase in cell death, and this DNA may also accumulate in the urine.
The circulating DNA from the bloodstream that passes into the urine can be isolated and used in many different applications in diagnostics. The DNA can be used for molecular diagnostics and prognosis, including cancer testing, prenatal diagnosis, and transplantation monitoring (U.S. Pat. Nos. 6,492,144 and 6,251,638). There are a number of advantages to using the DNA found in bodily fluids, such as urine, saliva, serum, tears, sweat, cerebral spinal fluid and plasma, for diagnostics, for example many of these fluids can be collected by non-invasive methods.
Classical methods of isolating DNA from urine generally require a large amount of urine, and result in the isolation of only a small amount of DNA. One of the most traditional methods of isolating urine DNA involves centrifuging 10 mL of urine, resuspending the pellet in a small volume of the urine supernatant, adding lysis solution that contains proteinase K, and incubating for up to 4 hours at 55° C. After incubation, the sample is extracted once with TE phenol, twice with phenol:chloroform:isoamyl alcohol and once with chloroform:isoamyl alcohol. Finally, sodium acetate is added to the sample, it is precipitated with ethanol overnight, washed with 70% ethanol the next day and resuspended in TE buffer. This method is long, tedious and often results in only trace amounts of DNA being isolated (Yokota et al., 1998).
In another published method for isolating DNA from urine (Su et al., 2004), urine samples are added to 1.5 volumes of 6M guanidine thiocyanate and mixed by inversion. A resin is then added to the sample and incubated for 2 hours. The resin-DNA complex is then centrifuged, transferred to a mini-column, washed with a buffer and the DNA eluted with water. Again, this method requires large amounts of urine and results in only small amounts of DNA being isolated.
Much of the DNA present in urine is protein-bound, while only a small portion of the urine DNA is free in the urine. With the classical approaches, including those outlined above, only the free DNA is being isolated. Since free DNA constitutes only a small portion of the total DNA present in urine, the yields using these classical approaches will be low and consequently large volumes of urine will be required to isolate sufficient urine DNA to be used in downstream applications. In other words, only a sub-population of the total DNA from urine is isolated. Given that a significant amount of the DNA present in urine is protein-bound, in order to isolate total DNA from urine one must isolate both the free DNA and the proteins present in the urine, and then release the bound DNA from these proteins. The existing methods rely on protein elimination and DNA retention in the first and subsequent steps, inevitably resulting in a loss of a significant amount of protein bound DNA.
Thus, there exists a need for a novel method to isolate pure and biologically active total DNA from small amounts of bodily fluids, including urine, serum, saliva, tears, sweat, cerebral spinal fluid and plasma.